Production and use of magnetic porous inorganic materials

ABSTRACT

Magnetic porous inorganic siliceous materials having a particle size of about 1 to about 200 microns useful as solid supports in various chromatography, immunoassays, synthesis and other separation and purification procedures is disclosed.

This application is a continuation-in-part of application Ser. No.08/307,307, now U.S. Pat No. 5,601,979 filed Sep. 16, 1994, which is acontinuation-in-part of application Ser. No. 07/794,910 filed Nov. 20,1991.

FIELD OF INVENTION

This invention provides porous inorganic, magnetic materials useful inbiochemical synthesis, assay, purification and separation procedures.More particularly the invention relates to magnetic siliceous inorganicmaterials such as controlled pore glass (CPG), porous silica gel, andporous ceramic products, to methods for the preparation of suchproducts, and to various uses for such products. The invention relatesto the surface modification of such products by, e.g., physicallyadsorbed or chemically immobilized biological molecules, and topractical applications thereof.

BACKGROUND OF THE INVENTION

Porous inorganic siliceous materials including glass, ceramics, andsilica gel are used as solid supports in chromatography, immunoassays,synthesis and other separation and purification procedures.

Gravitational and centrifuged separation of such porous materials fromthe surrounding medium when used in batch procedures such asimmunoassays, is inefficient and time consuming. Centrifugal separationsalso require expensive and energy consuming apparatus.

Separation of magnetic solid supports is relatively easy and simple,especially for multiple, small aliquots of the kind frequentlyencountered in sample preparation and immunoassay procedures. Agitationof magnetic solid supports is readily accomplished by on and offswitching of magnetic fields located at opposite sides of a container orsimply shaken by hand. Non-porous metal oxide magnetic particles andmagnetic polystyrene beads lack surface area necessary to provide highbinding capacity.

Although there are quite a number of magnetic materials commerciallyavailable or reported in the literature, such as: iron oxide particlesof U.S. Pat. Nos. 4,554,088 and 3,917,538; nickel oxide particles inBiotec. and Bioengr. XIX:101-124 (1977); agarose-polyaldehyde beadcontaining magnetic particles of U.S. Pat. No. 4,732,811. Commercialproducts such as: "DYNABEADS" (magnetic polystyrene bead); "MAGNOGEL 44"(magnetic polyacrylamide-agarose); "ENZACRY" (poly-m-diaminobenzene ofiron oxide) reported in Clin. Chim. Acta. 69:387-396 (1976). Other typesof magnetic particles reported in the literature include: cellulosecontaining ferric oxide, Clin. Chem. 26:1281-1284 (1980) and albuminmagnetic microspheres, Ovadia, et al. J. Immunol. Methods 53:109-122(1982).

SUMMARY OF THE INVENTION

This invention provides a novel and simple method for making porousinorganic magnetic materials including any glass, silica gel or alumina,useful, e.g., in the separation of biochemical moieties or biologicalmolecules or fragments thereof from a surrounding medium, in thesynthesis of peptides and oligonucleotides, in the purification of mRNAor poly (dA) directly after synthesis and in DNA assay procedures invarious immunoassay procedures for enzyme immobilization and in samplepreparation.

The magnetic products of the invention have a pore diameter of fromabout 60 to about 6,000 Angstroms (A), preferably between about 300 A toabout 5,000 A. Specific pore volume, which is proportional to thesurface area for a given pore size is from about 0.5 to about 2.5 cc/gm,preferably from about 0.75 to about 1.5 cc/gm. Particle size is fromabout 1 to about 200 microns, preferably from about 5 to about 50microns.

This invention provides both ferromagnetic materials andsuperparamagnetic materials. The latter are preferred to precludemagnetic aggregation and to facilitate redispersion upon removal of amagnetic field.

This invention also includes porous inorganic magnetic materials,preferably siliceous materials, surface modified to provide functionalgroups such as amino, hydroxyl, carboxyl, epoxy, aldehyde, sulfhydryl,phenyl or long chain alkyl groups to facilitate the chemical and/orphysical attachment of biological molecules and other moieties, e.g.,enzymes, antibodies, oligopeptides, oligonucleotides, oligosaccharidesor cells. Surface modification to create such functionality may beaccomplished by coating with organic silanes. See, e.g., BondedStationary Phases in Chromatography, ed. by E. Grushka (1974). Alternatemethods for providing derivatized or functional group containingsurfaces on the magnetic products of this invention include U.S. Pat.Nos. 3,983,299 and 4,554,088.

It is known in the prior art to synthesize peptides by the addition ofadditional amino acid residues which are coupled to a first amino acidresidue bound to a solid support. An important aspect of this inventioncomprises utilizing as the solid support in such procedures magneticcontrolled pore glass having at least one amino acid residue covalentlyattached thereto. Prior art procedures for synthesizing peptides basedupon amino acid residues coupled to a solid support are useful pursuantto this aspect of the invention.

None of the prior art magnetic porous inorganic particles known toapplicant have the same practical range of pore diameter, narrow porediameter distribution, high pore volume, high surface area, surfacemodification versatility, solvent system compatibility and simplicity ofproduction as the products of this invention.

DEFINITIONS

The following definitions apply to this application:

The term "magnetic porous inorganic materials" is defined as any poroussiliceous inorganic materials such as porous glass, porous silica geland porous alumina, etc., which comprises magnetic materials eitherthrough physical adsorption or chemical binding.

The term "magnetic material" is defined as a transition metal oxidehaving ferrospinel structure and comprising trivalent and divalentcations of the same or different transitional metals, for example, ironoxide Fe₃ O₄.

The term "colloidal magnetic particles" is defined as finely dividedmagnetic materials of submicron size, usually 50-250 Angstrom. Suchparticles may be present in combination with a carrier liquid and asurfactant material and may remain dispersed throughout a carrier liquidmedium.

The term "superparamagnetism" is defined as the magnetic behaviorexhibited by the magnetic materials, which respond to a magnetic fieldwithout resultant permanent magnetization.

The term "solid phase sandwich type radioimmunoassay (RIA)" refers to animmunoassay in which a solid phase is first immobilized with an antibody(or antigen) and is then used to bind the targeted antigen (or antibody)in a sample. A second antibody (or antigen) labelled with radioactivematerials is then added to bind the antigen (or antibody) serving as asignal for the presence of the target antigen (or antibody). Theimmunocomplex formed on the solid phase would be like Ab-Ag-Ab* (orAg-Ab-Ag*), hence, a sandwich type immunoassay.

DESCRIPTION OF THE FIGURE

FIG. 1 is a scanning electron microscopic image of an MPG particle,30-40 microns in size having 3000 Å pores.

DETAILED DESCRIPTION OF THE INVENTION

The porous inorganic magnetic materials of the invention are produced byadding magnetic metallic particles such as iron oxide, preferably as anaqueous colloidal suspension to an aqueous slurry of CPG, siliceousmaterial such as silica gel, or alumina, agitation of the mixture,removal of excess magnetic particles, and drying the product. Aqueouscolloidal iron oxide is preferred.

The CPG, silica gel or alumina used in the process is selected to have apore diameter, pore volume and particle size to provide a final porousmagnetic product of the desired physical characteristics. Combinationwith, e.g., iron oxide, may reduce original pore volume by about 5% toabout 15%.

Controlled pore glass useful in this invention is commercially availablein a range of pore dimensions from CPG, Inc., 32 Pier Lane West,Fairfield, N.J. The production of controlled pore glass is described inU.S. Pat. Nos. 3,549,524 and 3,758,284.

Colloidal magnetic particles useful in the invention constitute fromabout 2% to 15% by volume of magnetic particles in liquid, preferablywater, suspension medium. Colloidal iron oxide is commercially availableas "Ferrofluid" (trademark) from Ferrofluidics Corp., 40 Siman Street,Nashua, N.H. Ferrofluids containing from about 1% to about 6% of ironoxide in water or organic phase such as perfluorinated polyether ordiester are useful in the practice of the invention. The production offerrofluid is described in U.S. Pat. Nos. 3,531,413 and 3,917,538.

Agitation of the mixture of porous inorganic material and colloidalmagnetic particles is appropriately accomplished by shaking or by anon-metallic mixer at room temperature for a time period of from about 3to 96 hours. Discoloration of, e.g., CPG, indicates adsorption orlodging of the colloidal magnetic particles within the pores of theinorganic material.

Removal of unbound colloidal magnetic particles may be accomplished bywashing with water followed by polar liquids. An appropriate washingsequence is water, 1.5M aqueous sodium chloride, acetone and methanol.Each wash step is continued until the supernatant is clear.

The final, washed, magnetic particles are filtered and dried, e.g.,overnight at 90° C. or at 120° C. for one hour or vacuum dried for sixhours. Depending on the pore diameter, the dry magnetic porous particlesappear light to dark brown in color and respond to a magnetic field. Ingeneral, materials of relatively small pore diameter which have a higherspecific surface area adsorb more colloidal magnetic particles and,hence, exhibit stronger magnetic properties than materials of largerpore diameter.

To provide functional groups for the binding of biological moietiesincluding cells and biomolecules. The magnetic porous particles may thenbe subjected to surface modification such as silanization. See, e.g.,Grusha, supra and U.S. Pat. Nos. 3,383,299 and 4,554,088. It alsosecures immobilization of the magnetic particle in the inorganicmaterial pores.

A general formula for the silicone compounds useful for silanization is:R-Si-X, where R represents an organic moiety with a terminal functionalgroup such as an amino, hydroxyl, epoxy, aldehyde, sulfhydryl, phenyl,long chain alkyl or other group that will chemically react or physicallyabsorb with the biological molecules and X may be a mono-, di-ortrialkoxy or halide group which will react with the silanol groups onthe surface of the inorganic material. The degree of silanization can bedemonstrated through quantitative analysis of the respective functionalgroups.

The preferred colloidal magnetic particles for use in this invention aresuperparamagnetic metal oxide. The size of the colloidal particles mayrange from 1 to 100 nm, preferably 5 to 50 nm (50 to 500 Angstroms (A)).Other superparamagnetic colloidal solutions are described in U.S. Pat.Nos. 3,215,572 and 4,554,088.

EXAMPLE I Preparation of Magnetic Porous Inorganic Material WithFerrofluid Colloidal Particles

5 gm of controlled pore glass (CPG, pore diameter of 3000 Angstrom,37-77 microns) was added to a 70 ml container containing 50 ml ofdeionized water. To the glass slurry, 1 ml of Ferrofluid colloidal ironoxide (Ferrofluidics Corp.) was added. The Ferrofluid contained 1 to 3%by volume superparamagnetic 100 A iron oxide particles in an aqueousmedium. The container was placed in the shaker and gently shaken for 24hours. The glass particles turned into dark brown color. ExcessiveFerrofluid was decanted off after the glass settling down. After fivewashes with water, one wash with 1.5M NaCl solution, three more waterwashes and three more methanol washes, the magnetic controlled porousglass (magnetic CPG) was then filtered and dried at 90° C. for eighthours. The final product was attracted by laboratory permanent magnet.

Physical characteristics of the magnetic controlled porous glass(magnetic CPG) product were checked by microscopic examination. Poremorphology was determined by porosimeter and surface area analyzer.Under the microscope, the appearance of the magnetic CPG was the same asthe regular porous glass except that the magnetic CPG particle was of auniform brown color. The porosity data for both before and after coatingmagnetic particles are listed in Table 1. Specific pore volume wasdecreased as expected, because part of the pore volume was occupied bythe colloidal iron oxide particles. The increase in the surface area isdue to the existence of colloidal particles.

                  TABLE 1                                                         ______________________________________                                        Porosity Data For Glass Particles Before and                                  After Coating with Magnetic Colloidal Particles                                                Before                                                                              After                                                                   Coating                                                                             Coating                                                ______________________________________                                        Mean pore dia. (A) 3000    3000                                               Specific pore vol. (cc/gm)                                                                       0.89    0.84                                               Pore diam. distribution (%)                                                                      8.4     6.9                                                Surface area (M2/gm)                                                                             7.4     8.97                                               Lot No.            11C24   081783-2                                           ______________________________________                                    

EXAMPLE II Preparation of Magnetic Silica Gel With Colloidal MagneticParticles (Magnetic Silica Gel)

5 grams of Daisogel, a silica gel product of Daiso Co., Inc., 10-5,Edobori 1-Chome Nishi-Ku, Osaka, Japan, having pore diameter of 1000Angstrom, 5 micron spherical bead was slurried in a 70 ml bottlecontaining 50 ml of tetrahydrofuran. To the silica gel slurry, 1 ml offerrofluid colloidal iron oxide was added. The ferrofluid contained 3 to6% by volume of superparamagnetic 100 A iron oxide particles in organicbase medium. The container was placed in the shaker and shaken for 24hours at room temperature. At the end of mixing time, the excesssolution was decanted off. The silica gel was then washed with 3×10 mlof tetrahydrofuran, 3×10 ml of ethyl acetate, 5×10 ml of methanol andfinally another 5×10 ml of deionized water. During each washing cycle, apermanent magnet was used to accelerate the settling down of themagnetic silica gel. The magnetic silica gel was then dried at 120° C.for 1 hour. The porosity data for both uncoated and magnetic colloidalparticle coated Daisogel are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                        Porosity Data for Silica Gel Particles                                        Before and After Coating with Colloidal Iron Oxide                                             Before  After                                                                 Coating Coating                                              ______________________________________                                        Mean pore diameter (Angstrom)                                                                    688       685                                              Specific pore volume (cc/gm)                                                                     .95       .85                                              Pore diam. distribution (%)                                                                      27.3      24.7                                             Surface area (M.sup.2 /gm)                                                                       67.1      60.6                                             Lot No.            DS-GEL05  MSIL1005                                         ______________________________________                                    

EXAMPLE III Preparation of Magnetic Porous Inorganic Materials WithColloidal Iron Oxide Particles

Colloidal iron oxide was prepared by the method of U.S. Pat. No.4,554,088 with some modification: A 20 ml of 2:1 molar ratio of FeCl₂/FeCl₃, solution was mixed with equal volume of 4.5M sodium hydroxide toform a crystalline precipitate of superparamagnetic ion oxide, having aparticle size diameter of 0.1 to 1.5 microns. For the purposes of thisinvention, such particle size was too large to produce magnetic porousparticles. To obtain the appropriate colloidal size of iron oxideparticles, the concentration of ferrous/ferric chloride was diluted atleast 10 fold, the mixing of iron chloride solution and sodium hydroxidewas done in a ultrasonic bath for at least two hours, and the pH of theprecipitate solution was adjusted to about 7.5. The particle size wasmonitored by microscopic observation or by light scattering technique.Aggregation, if any, found among the colloidal particles was washed awayin the course of the porous material coating procedure. The final ironoxide particle size was about 200 Angstrom to about 500 Angstrom.

2 gm of controlled pore glass (CPG, pore diameter of 1000 Angstrom,77-125 microns (CPG, Inc.)) was mixed with 10 ml colloidal iron oxide(50 vol. % precipitate). The slurry was shaken gently in the shaker for24 hours. Excessive colloidal iron oxide was decanted off, and glassslurry was exhaustively washed with water until the supernate becameclear. The glass was then washed with methanol, filtered and dried inthe oven at 90° C. for eight hours. The final product was brown in colorand attracted by a permanent magnet.

EXAMPLE IV Preparation of Magnetic Amino Controlled Pore Glass (MagneticAmino CPG)

The product of Example I was further dried under vacuum at roomtemperature for two hours. 5 gm of the dried magnetic CPG was placed ina three neck round bottom flask. 150 ml of 10%gamma-aminopropyltri-methoxysilane in dry toluene was added to theflask. The slurry was gently stirred under refluxing condition for 24hours. The glass was then washed with methanol for five times to removeexcessive silane. The settling process could be sped up by placing acircular magnet under the container. The glass was then filtered andbaked in the oven at 90° C. for eight hours. The magnetic amino glass(magnetic amino CPG) was quantified by titration and found to have 35.5micromole amino groups per gram of solid.

EXAMPLE V

The product of Example IV is utilized as a solid support in any knownprocedure for the synthesizing of a peptide by the coupling ofadditional amino acid residues to a supported amino acid residue.

EXAMPLE VI Preparation of Magnetic Epoxide Controlled Pore Glass(Magnetic Epoxide CPG)

5 gm of dried magnetic CPG prepared as described by Example I was placedin a three neck round bottom flask. 150 ml of 10%3-glycidoxypropyltrimethoxysilane in dry toluene was added to the flask.The slurry was gently stirred under refluxing condition for 24 hours.The magnetic CPG was then washed with methanol and acetone to removeexcessive silane. The magnetic CPG glass was then filtered and baked inthe oven at 100° C. for 16 hours. The epoxide group was quantified bytitration and found to have 42 micromoles per gram of solid.

EXAMPLE VII Coupling of Anti-HBsAg to Magnetic Amino-Controlled PoreGlass (Magnetic Amino-CPG)

One gram of magnetic amino glass (magnetic amino-CPG) prepared fromExample IV was added to a bottle containing 30 ml of 10% aqueousglutaraldehyde at pH-7.0. The slurry was shaken gently in the shaker forone half hour at room temperature. 30 mg of sodium borohydride was thenadded and the slurry was then shaken in an ice-water bath for threehours. At the end of the reaction, the glass was washed with phosphatebuffer thoroughly. The settling of the glass particles was acceleratedby using a magnetic field. The amino groups on the surface of the glasswere thus converted to aldehyde moieties.

9.9 ml (1 mg/ml protein conc.) of crude goat anti-human hepatitis Bsurface antigen (anti-HBsAg) antibody solution (Electro-NucleonicsLaboratory, Inc.) was added to 1 gm of magnetic aldehyde glass and 12 mgof sodium borohydride. 0.1M sodium carbonate of pH=9.5 was used toadjust the pH of the mixture to 8.5. The slurry was shaken in therefrigerator for 24 hours. The antibody coupled particles was thenwashed three times with 0.1M sodium phosphate buffer, pH=7.5 (fivetimes). To block any active sites from residue silanol, amino oraldehyde groups, 5 ml (2 mg/ml) human serum albumin solution was treatedwith the magnetic antibody coated glass particles for three more hours.The magnetic antibody coated glass (magnetic antibody-CPG) slurry wasthen washed with phosphate buffered saline (PBS) three time, 1M NaClonce, and back to PBS three more times. The particles were then storedin the refrigerator for use in immunoassay procedures.

EXAMPLE VIII Magnetic Antibody Coated Controlled Pore Glass (MagneticAntibody-CPG) For Sandwich Type Radioimmunoassay (RIA) For HumanHepatitis B Surface Antigen HBsAg

200 microliter of four negative and three positive serum standardscontaining deactivated human hepatitis B surface antigen were applied toeach Riasure assay tube ("Riasure" is the trademark for radioimmunoassayfor human hepatitis B surface antigen, produced by Electro-NucleonicsLaboratory, Inc.) containing one tablet form of CPG powder (whichdisintegrated back into powder form in the serum sample) or 10microliter of the magnetic antibody CPG slurry, prepared from ExampleVII in working buffer (1:1 vol. %). After one hour of incubation at 25°C., both glass slurrys were washed five times with supplied phosphatebuffer saline (PBS). The washing cycle for non-magnetic glass particleswere 60 seconds stirring and 90 seconds settling; for magnetic particle,the washing cycles had been cut down to 60 seconds stirring and 20seconds of settling with the help of an external magnetic field on theside. After five washing cycles, 100 microliter of radioactive iodine(I¹²⁵) labelled goat anti-hepatitis B surface antibody (I¹²⁵ anti-HBsAg)was then added to each assay tube. After another hour incubation at 25°C., the glass particles were again subjected to five PBS (phosphatebuffered saline) washing cycles prior to radiation count. The resultsobtained from RIA are presented in the following Table 3.

                  TABLE 3                                                         ______________________________________                                        Radioimmunoassay For Hepatitis B Surface                                      Antigen With Regular And Magnetic Glass Particles                                         Count Per Minute (CPM)                                            Samples       Regular CPG                                                                             Magnetic CPG                                          ______________________________________                                        Negative      169       200                                                   Negative      142       271                                                   Negative      196       347                                                   Negative      161       233                                                   Positive      25589     39026                                                 Positive      22551     33243                                                 Positive      25909     36257                                                 Ratio of P/N  147.8     137.5                                                 ______________________________________                                    

EXAMPLE IX Preparation of Magnetic Nucleoside CPG

Magnetic dT-CPG is prepared to demonstrate the production of magneticnucleoside CPGs. Deoxythymidine (dT) is used in this example. dA, dC anddG CPG products are produced in like manner.

5 gram of dried magnetic epoxide CPG prepared in Example VI was placedin a 100 ml round bottom flask. To the dried glass powder, 5 gram of1,6-hexanediamine in 50 ml of dried methanol was added. The slurry wasstirred gently at room temperature for three hours. At the end of thereaction, the glass was washed with methanol, 0.05 m sodium acetatebuffer of pH 5.5, then deionized water, then final methanol wash beforeit was filtered and dried. The magnetic long chain amino glass (MagneticLong Chain Amino CPG) was found to have 35 micromole of primary amineper gram of solid. One gram of this magnetic long chain amino glass, 160mg of DMTr-deoxythy-midine succinic acid, 0.160 ml1,3-diisopropyl-carbodiimide, 2.2 mg 4-dimethylaminopyridine, 1 mlpyridine and 4 ml N,N-dimethylformamide were mixed together in a 8 mlamber vial. The vial was placed on an orbitory shaker for shaking 24hours at room temperature. At the end of the reaction, the glass wascapped with 0.1 ml acetic anhydride for three hours followed byquenching the excessive anhydride with 0.2 ml dried methanol in ice-bathfor another three hours. The magnetic DMTr-thymidine glass (magneticDMTr-dT-CPG) was then washed with N,N-dimethylformamide, methanol anddichloromethane before subjected to vacuum drying. The glass wasquantified by cleaving the DMTr (dimethoxytritryl-) moiety from theglass with 3% p-toluenesulfonic acid in acetonitile and measure itsabsorbance at 504 nm. The DMTr groups were found to be 23 micromoles pergram of solid.

EXAMPLE X Synthesis of 20-Mer Oligonucleotide With MagneticDMTr-deoxythymidine CPG (Magnetic DMTr-dT CPG)

10 mg of the magnetic dT-CPG from Example IX was packed in a DNAreaction column. The column was placed in the DNA synthesizer of model381A manufactured by Applied Biosystems, Inc. (ABI). β-cyanoethylphosphoramidites and other synthetic reagents for synthesis wereacquired from ABI. A 20 mer oligonucleotide of the following sequencewas synthesized, i.e., AGA/CAG/TCT/GAT/CTC/GAT/CT (SEQ ID NO. 1). TheDMTr groups, which were removed in each synthesis cycle, were collectedand measured at 504 nm to check for coupling efficiency. The 20 merswere then cleaved off from the solid phase and subjected to HPLCanalysis. The results were found to be the same as those generated fromregular non-magnetic glass particles.

EXAMPLE XI Synthesis of Non-Clearable 25-Mer Oligonucleotide WithMagnetic Controlled Pores Glass (Magnetic Oligonucleotide CPG)

1 gram of magnetic epoxy CPG from Example VI was hydrolized in 10 ml ofacidic aqueous solution at pH=4.0 (adjusted with hydrochloric acid) andat 40° C. for two hours. At the end of reaction, the magnetic CPG waswashed five time with 50 ml deionized water, because the epoxy group wasconverted into dihydroxyl group. This material was designed as magneticglyceryl glass (magnetic glyceryl-CPG). 10 mg of this material was thenpacked in a DNA synthesis column. The column was placed in the automaticDNA synthesizer of model 381A manufactured by Applied Biosystems Inc.Beta-cyanoethyl phosphoramidites and other reagents for synthesis wereacquired from the same company. A 25-mer of deoxythymidineoligonucleotide of the following sequence was synthesized, i.e.,TTT/TTT/TTT/TTT/TTT/TTT/TTT/TTT/T (SEQ ID NO. 2). The magnetic glasspowder bearing the 25-mer was then subjected to the treatment ofammonium hydroxide to remove the phosphate protective groups. Due to themore stable phosphodiester linkage between the 25-mer oligonucleotidechain and the glass, a large fraction of the oligonucleotides remainedcovalently linked to the magnetic glass as confirmed by the DMTr groupsand by the capability of the product to hybridize poly(dA)₁₂oligonucleotides. Products bearing the 25-mer are useful to purify mRNAand poly(dA) immediately after synthesis. It is also useful in DNAassays. The magnetic glass with non-cleavable synthetic oligonucleotidesis also useful in DNA assay.

EXAMPLE XII Preparation of Protein A Coated Magnetic Controlled PoreGlass (Magnetic Protein A CPG) Useful as an Antibody Adsorbent

One gram of the product of example V (magnetic epoxy CPG) was placed ina vial containing 5 ml of 0.1 m sodium periodate aqueous solution. Thevial was placed on a shaker and shook for 1 hour. At the end ofreaction, the glass was washed with 5×5 ml deionized water. 15 mg ofProtein A was dissolved in 5 ml of 0.01M phosphate buffer of pH=7.2 andadded to the glass. The vial was shaken gently in the refrigerator for24 hours. At the end of coupling reaction, 0.02% (wt %) of sodiumborohydride was added to the mixture, and the reaction was allowed toproceed for another two hours. pH was adjusted to around pH=8.5 to 9.0with dilute hydrochloric acid or sodium hydroxide if necessary. At theend of the reaction, the glass was washed 5×10 ml of phosphate buffer.The product was magnetic glass coated with Protein A.

200 mg of the Protein A magnetic glass was placed in a 8 ml vial whichcontained 5 ml of 10 mg goat anti-BSA (bovine serum albumin) antibody in0.05M phosphate buffer +0.15M sodium chloride of pH=7.5. The vial wasthen shook gently in the shaker for one half hour at room temperature.The glass was then washed with 5×5 ml of the loading buffer to removethe excess or unbound proteins. To elute the absorbed antibody from theProtein A magnetic glass, 3×1 ml of 0.1M glycine/HCl buffer of pH=2.0was used. The washing buffers were pooled together and the proteinconcentration was measured by Lowry's method at 280 nm. The Protein Amagnetic glass was thus found to have a binding capacity of 8 mg goatanti-BSA (bovine serum albumin) antibody per gram of magnetic Protein ACPG.

EXAMPLE XIII Hydroxyl Functionalization of MPG

5 grams of magnetic epoxide MPG prepared in Example VI was placed in a150 ml 3 necks round bottle flask. To the magnetic epoxide CPGparticles, 100 ml of 0.2M sodium periodate (NaIO₄) pH 2.2 (titrated with3M periodic acid was added. The slurry was stirred gently at 40° C. forsix hours. At the end of the reaction, the particles were washed with10×10 ml deionized water. 500 mg of sodium cyanoborohydride was added tothe 100 ml MPG slurry. The reaction was allowed to proceed for threehours. Then the particles were washed with 7×100 ml deionized water,1×100 ml acetone and filtered. The glass was dried under vacuumovernight. Quantification by titration demonstrated 28 umole of hydroxylgroups per gram.

EXAMPLE XIV Preparation of Streptavidin-MPG With Glyceryl-MPG

1 gm dry Glyceryl MPG (MGLY) prepared in Example XIII was wetted with 45ml deionized water and sonicated for a few seconds to ensure noaggregate in the suspension. The wet magnetic particles were activatedwith 0.2M sodium meta-periodate for 1.5 hour at room temperature. At theend of the reaction, the particles were washed ten times with 40 mldeionized water. The Streptavidin solution was prepared by adding 200 mgstreptavidin to 10 ml of 0.1M phosphate buffer, pH=7.40 (couplingbuffer). The protein solution was added along with 0.1 g of sodiumcyanoborohydride to the activated MGLY particles. The particles andprotein solution was allowed to tumble overnight at room temperature ona low speed rotator. Excess protein solution was removed and theparticles were washed once with 40 ml coupling buffer. The efficiency ofprotein coupling was determined by the difference of two proteinconcentrations (reading at 280 nm in a spectrophotometer before andafter the reaction). In order to cap the unreacted site, 10 ml ofcoupling buffer containing 3.76 gm of glycine and 0.1 gm sodiumcyanoborohydride was added to the particles. The reaction mixture wasagain allowed to tumble for another three hours at room temperature. Atthe end of the reaction, the particles were washed once with 40 mlcoupling buffer, three times with 40 ml PBS (0.01M phosphate+1.5M NaCl,pH=7.40), and three times with 40 ml deionized water and twice with 40ml of storage buffer (PBS+) 0.1% bovine serum albumin+0.02% sodiumazide).

EXAMPLE XV Preparation of Hydrazide MPG

This Example demonstrates the production of a magnetic pore glass (MPG)derivative, the surface of which is modified to possess hydrazinegroups. This aspect of the invention is important because a moleculewith an aldehyde group reacts directly with hydrazine on the solidsurface to form a stable hydrazone bond. Unlike the C=N bond (a Schiffbase) formed by the aldehyde and an amine, it requires no furtherreduction step. The major application for this product is for antibodycoating on MPG.

One gram of dried Epoxy-MPG prepared in Example V was wetted with 45 mldeionized water and sonicated for a few seconds to insure no aggregatein the suspension. The particles were magnetically separated and thesupernatant discarded. 40 ml of 0.2M sodium meta-periodate, pH=2.2(titrated with 3M periodic acid) was added to the wet magneticparticles. The oxidation reaction was allowed to continue for six hoursat 40° C. At the end of the reaction, the activated epoxy MPG particleswere washed ten times with 40 ml deionized water. Hydrazide solution wasprepared by adding 3.48 g of adipic acid dihydrazide in 30 ml of 0.01Macetate buffer, pH=4.00. The hydrazide solution was added along with 0.1g of sodium cyanoborohydride to the activated epoxy EPG particles. Theparticles and hydrazide solution was allowed to tumble overnight at 40°C. on a low speed rotator. At the end of reaction, after magneticallyseparating the particles, the excess hydrazide solution was discarded.The hydrazide coated particles were then washed once with 40 ml acetatebuffer and five more times with 40 ml of deionized water. The capacityof the hydrazine was determined by the titration and was found to be120μ mole/gm.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: Nucleotide                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Unknown                                                         (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      AGACAGTCTGATCTCGATCT20                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25                                                                (B) TYPE: Nucleotide                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Unknown                                                         (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TTTTTTTTTTTTTTTTTTTTTTTTT25                                                   __________________________________________________________________________

I claim:
 1. Particulate paramagnetic porous glass comprising non-magnetic controlled porous glass having paramagnetic particles bonded to or adsorbed on its surface and in its pores, said non-paramagnetic controlled porous glass having(i) a particle size of from about 1 to about 200 microns; (ii) an original pore diameter of about 60 to about 6,000 Angstroms; (iii) an original pore volume of about 0.5 to about 2.5 cc/gm; andwherein said non-magnetic controlled porous glass is rendered paramagnetic.
 2. In a process for the synthesis of an oligonucleotide in which a nucleoside bound to a solid support is utilized, the improvement which comprises utilizing as said solid support a paramagnetic, porous glass as defined by claim
 1. 3. A process as defined by claim 2 in which said paramagnetic porous inorganic material is paramagnetic controlled pore glass.
 4. Particulate paramagnetic porous glass comprising non-magnetic controlled porous glass having paramagnetic particles bonded to or adsorbed on its surface and in its pores, said non-paramagnetic controlled porous glass having(i) a particle size of from about 1 to about 200 microns; (ii) an original pore diameter of about 60 to about 6,000 Angstroms; (iii) an original pore volume of about 0.5 to about 2.5 cc/gm; and (iv) a surface bearing a hydrazine moiety wherein said non-magnetic controlled porous glass is rendered paramagnetic.
 5. Particulate paramagnetic porous glass as defined by claim 1 or claim 4 in which said paramagnetic particles are paramagnetic iron oxide particles.
 6. Particulate paramagnetic porous glass as defined by claim 1 or claim 4 in which said paramagnetic particles are colloidal paramagnetic iron oxide particles.
 7. Particulate paramagnetic porous glass as defined by claim 1 or claim 4 in which the original pore volume is reduced by about 5% to about 15% by paramagnetic iron oxide particles.
 8. Particulate paramagnetic porous glass comprising non-magnetic controlled porous glass having paramagnetic particles bonded to or adsorbed on its surface and in its pores, said non-paramagnetic controlled porous glass having(i) a particle size of from about 1 to about 200 microns; (ii) pores having a pore diameter of about 60 to about 6,000 Angstroms and an original volume of about 0.5 to 2.5 cc/gm; (iii) a surface to which a biological molecule or a cell may be attached; andwherein said non-magnetic controlled porous glass is rendered paramagnetic.
 9. A particulate paramagnetic porous glass as defined by claim 8 in which said biological molecule is an enzyme, an antibody, an antigen, a nucleoside, an oligonucleotide, a peptide or an oligosaccharide.
 10. Particulate paramagnetic porous glass comprising non-magnetic controlled porous glass having paramagnetic particles bonded to or adsorbed on its surface and in its pores, said non-paramagnetic controlled porous glass having(i) a particle size of from about 1 to about 200 microns; (ii) pores having a diameter of about 60 to about 6,000 Angstroms and a pore volume of about 0.5 to 2.5 cc/gm; and (iii) an external surface bearing functional groups to chemically bind biological molecules or cells theretowherein said non-magnetic controlled porous glass is rendered paramagnetic.
 11. Particulate paramagnetic porous glass as defined by claim 10 in which said functional groups are an amino, hydroxyl, carboxyl, epoxy, aldehyde, phenyl or alkyl groups.
 12. A biological molecule or a cell bound to a paramagnetic controlled pore glass support as defined by claim
 1. 13. The invention as defined by claim 12 in which said biological molecule is an antibody, an antigen, a peptide, a nucleoside, an oligonucleotide or an oligosaccharide.
 14. A process for producing paramagnetic porous glass which comprises:(i) mixing porous glass particles having a particle size of from about 1 to about 200 microns, and pores having a diameter of from about 60 to about 6,000 Angstroms and a pore volume of from about 0.5 to about 2.5 cc/gm, with colloidal paramagnetic iron oxide particles about 50 to 500 Angstroms in size, wherein said mixing yields paramagnetic porous glass having said paramagnetic iron oxide particles contained in the pores and adsorbed on the pore surfaces; and (ii) thereafter separating, washing and drying said paramagnetic porous glass particles.
 15. A process as defined by claim 14 in which said washing in step (ii) includes an initial washing with water followed by washing with another polar liquid.
 16. A process as defined by claim 14 in which said washing in step (ii) includes an initial washing with water followed by washing with aqueous sodium chloride, acetone or methanol.
 17. A process as defined by claim 14 in which said drying in step (ii) is accomplished at a temperature of from about 90° C. to 100° C. for one hour or by overnight vacuum drying.
 18. In a process for the synthesis of an oligonucleotide in which a nucleoside bound to a solid support is utilized, the improvement which comprises utilizing as said solid support paramagnetic porous glass having(i) a particle size of from about 1 to about 200 microns; (ii) an original pore diameter of about 60 to about 6,000 Angstroms; (iii) an original pore volume of about 0.5 to about 2.5 cc/gm; and (iv) paramagnetic particles contained in and adsorbed on the surfaces of said pores. 